Successful secretion of proteins has been accomplished both with prokaryotic and eukaryotic hosts. The most prominent examples are bacteria like Escherichia coli, yeasts like Saccharomyces cerevisiae, Pichia pastoris or Hansenula polymorpha, filamentous fungi like Aspergillus awamori or Trichoderma reesei, or mammalian cells like e.g. CHO cells. While the secretion of some proteins is readily achieved at high rates, many other proteins are only secreted at comparatively low levels.
The heterologous expression of a gene in a host organism requires a vector allowing stable transformation of the host organism. This vector or expression cassette has to provide the gene with a functional promoter adjacent to the 5′ end of the coding sequence. The transcription is thereby regulated and initiated by this promoter sequence.
The secretory pathway typically starts by translocation of transmembrane polypeptides and polypeptides intended for secretion into the lumen of the endoplasmic reticulum (ER). For that purpose, these proteins possess an amino-terminal precursing sequence, also called “leader”, comprising or consisting of a signal peptide and an optional secretion leader pro-peptide. The signal peptide typically consists of 13 to 36 rather hydrophobic amino acids. Signal peptides have a common structure: a short, positively charged amino-terminal region (n-region); a central hydrophobic region (h-region); and a more polar carboxy-terminal region (c-region) containing the site that is cleaved by the signal peptidase. On the ER luminal side the signal peptide is cleaved off by a signal peptidase. After successful folding of the nascent polypeptide by ER resident chaperones and foldases, the protein is further directed to exit the ER. This process may be supported by the presence of an N-terminal pro-sequence, as it is present e.g. at the precursor of the S. cerevisiae mating factor alfa (MFα). The protein is then transported to the Golgi network and finally to the plasma membrane for secretion into the supernatant. The leader pro-peptide is cleaved off the protein by (presumably) Golgi-resident proteases such as Kex2 protease of S. cerevisiae. 
As the majority of yeasts do not secrete large amounts of endogenous proteins, and their extracellular proteomes are not extensively characterized so far, the number of available secretion sequences for the use in yeasts is limited. Therefore the fusion of the target protein to the mating factor alfa leader peptide (MFα) from S. cerevisiae was employed to drive secretory expression in many yeast species (including Pichia, Kluyveromces, Zygosaccharomyces). Unfortunately the proteolytic processing of the MFα by Kex2 protease often yields heterogeneous N-terminal amino acid residues in the product.
EP324274B1 describes improved expression and secretion of heterologous proteins in yeast employing truncated S. cerevisiae alfa-factor leader sequences, EP301669B1 the Kluyveromyces alfa-factor leader sequence for secretion of heterologous proteins.
Alternatively, the signal peptides of S. cerevisiae phosphatase (PHO5, DK3614), S. cerevisiae sucrose invertase (SUC, WO84/01153), and yeast aspartic protease 3 (YAP3, EP792367B1) were used for secretory expression in yeast. EPO438200 (A1) discloses the signal peptide sequence of S. cerevisiae SUC2 of for the expression in P. pastoris. 
U.S. Pat. No. 5,268,273 describes a P. pastoris acid phosphatase (PHO1) signal sequence, in most cases weaker than MFα.
U.S. Pat. No. 7,741,075 describes a P. pastoris PIR1 secretion signal peptide for recombinant protein expression and Pichia pastoris PIR1 and PIR2 anchor domain peptides for recombinant surface display.
Khasa et al. 2011 (Yeast. 28(3):213-26) describe the isolation of Pichia pastoris PIR genes and their utilization for cell surface display and recombinant protein secretion, in particular recombinant protein secretion in P. pastoris, utilizing the pre-pro signal of PpPir1p protein, without a comparison to MFα.
WO2011073367A1 and Kottmeier et al. 2011 (Applied Microbiology and Biotechnology. 91:1, 133-141) describe a hydrophobin signal sequence which mediates efficient secretion of recombinant proteins in Pichia pastoris, in particular the use of the pre-sequence or pro-sequence of Trichoderma reesei hydrophobin for secretion of eGFP in P. pastoris. 
In the course of P. pastoris genome sequencing 54 different sequences were listed as predicted signal peptides which include a cleavage site to provide for the secretion of proteins. (De Schutter et al. Nature Biotechnology doi: 10.1038/nbt. 1544 2009).
US2011/0021378A1 describes a set of 54 P. pastoris genes identified that contain a signal sequence, among them residues 1-21 of SEQ ID 8 as listed in US2011/0021378A1, again including a cleavage site to provide for the secretion of proteins.
EP2258855A1 describes regulatory sequences of a P. pastoris derived expression system, wherein a signal and leader sequence of the P. pastoris Epx1 protein is used as a 57 amino acid precursing sequence to facilitate expression and secretion of a protein of interest POI. A cleavage site for a signal peptidase is predicted to be following the signal sequence, i.e. between position 20 and 21, indicated as a hyphen in the sequence of the cleavage site: VSA-AP (SEQ ID 6).
It is desirable to provide alternative regulatory elements suitable for expressing a POI in a recombinant eukaryotic host cell, and methods to produce secreted proteins in eukaryotic cells which are simple and efficient, and could preferably lead to a homogeneous N-terminus of the POI.